At least six imprinted genes (p57Kip2, Kvlqt1, Mash2, Ins2, Igf2, and H19) have been mapped to a one megabase cluster at the distal end of mouse chromosome 7. The imprinted expression and physical organization of these genes is conserved in humans where they map to chromosome 11p15.5. Disruption of the normal imprinted expression of these genes is associated with prenatal lethality in mice and with Beckwith-Wiedemann Syndrome (BWS) and with several tumors in humans. In addition, the most frequent genetic defect associated with long QT syndrome maps to this region in humans. Finally, tissue culture studies strongly suggest the existence of a novel gene with tumor suppressor activity in this region. Our goals are to understand the molecular and genetic mechanisms underlying the allele restricted expression of these genes, to identify novel imprinted genes in the region, and to develop mouse models for the human diseases associated with misexpression of these imprinted genes.We have used molecular approaches to identify P1 and Bacterial Artificial Chromosome (BAC) clones that span the approximately 1 megabase imprinted region of distal mouse. Using exon trap and direct sequencing we have identified several potential genes. One gene so identified is the mouse Kvlqt1 gene, previously identified in human genetic studies as the gene responsible for the majority of cases of long QT syndrome. Maternally associated translocations in human KVLQT1 are associated with BWS. We have completed extensive analysis of the expression pattern of mouse Kvlqt1 showing that it is imprinted in a developmentally regulated fashion: expression in the early embryo is from the maternal chromosome but is biallelic in new born pups. These findings help to explain the paradoxical association of the gene with both long QT syndrome, inherited as a dominant mutation with no parent-of-origin effect, and also with BWS which shows complete parent of origin bias. To further elucidate the genetics of these diseases we have generated null and point mutations at the Kvlqt1 locus in embryonic stem cells have used blastocyst injection to generate mice carrying these mutations. Mice homozygous for null alleles of Kvlqt1 show bilateral deafness and severe balance disorders. Histological characterization of inner ear development in mutant mice is currently underway to determine the basis for this phenotype. In addition we have begun characterization of the heart physiology in these mice by ECG analysis in vivo and ex vivo and by analysis of the electrophysiology of single cardiac myocytes. Preliminary analysis indicates that deletion of the Kvlqt1 gene activity results in extended QT intervals in mice. This extension is exacerbated by stress and, interestingly, shows a strong gender bias.We are using molecular genetic approaches to characterize the mechanisms of imprinting concentrating on coordinate regulation of the H19 and Igf2 genes. H19 is expressed only from the maternal chromosome while Igf2 is expressed only paternally. Using transgenic mice we have identified elements required for silencing of the paternal copies of H19 transgenes. These mice identify two crucial elements: one element upstream of -0.7 kilobases and a second within H19s exon 1. We have generated mice carrying mutations that allow us to delete these regions separately and in a temporally controlled manner. We have shown that both Igf2 and H19 share a common imprinting control region. That is, the element just upstream of the H19 promoter is required for silencing of the paternal H19 and of the maternal Igf2 alleles. However, the molecular mechanisms for silencing of these genes is distinct. That is, the times during development when this cis acting element must be present to establish imprinting are distinct for the two genes. Further, our studies suggest that the imprinting element acts as a boundary, restricting access of enhancer elements to promoters on the other side of the boundary. We are currently testing this model using transgenic mice.